miR-346 was identified as an activator of Androgen Receptor (AR) signalling that associates with DNA damage response (DDR)-linked transcripts in prostate cancer (PC). We sought to delineate the impact of miR-346 on DNA damage, and its potential as a therapeutic agent.
RNA-IP, RNA-seq, RNA-ISH, DNA fibre assays, in vivo xenograft studies and bioinformatics approaches were used alongside a novel method for amplification-free, single nucleotide-resolution genome-wide mapping of DNA breaks (INDUCE-seq).
miR-346 induces rapid and extensive DNA damage in PC cells - the first report of microRNA-induced DNA damage. Mechanistically, this is achieved through transcriptional hyperactivation, R-loop formation and replication stress, leading to checkpoint activation and cell cycle arrest. miR-346 also interacts with genome-protective lncRNA NORAD to disrupt its interaction with PUM2, leading to PUM2 stabilisation and its increased turnover of DNA damage response (DDR) transcripts. Confirming clinical relevance, NORAD expression and activity strongly correlate with poor PC clinical outcomes and increased DDR in biopsy RNA-seq studies. In contrast, miR-346 is associated with improved PC survival. INDUCE-seq reveals that miR-346-induced DSBs occur preferentially at binding sites of the most highly-transcriptionally active transcription factors in PC cells, including c-Myc, FOXA1, HOXB13, NKX3.1, and importantly, AR, resulting in target transcript downregulation. Further, RNA-seq reveals widespread miR-346 and shNORAD dysregulation of DNA damage, replication and cell cycle processes. NORAD drives target-directed miR decay (TDMD) of miR-346 as a novel genome protection mechanism: NORAD silencing increases mature miR-346 levels by several thousand-fold, and WT but not TDMD-mutant NORAD rescues miR-346-induced DNA damage. Importantly, miR-346 sensitises PC cells to DNA-damaging drugs including PARP inhibitor and chemotherapy, and induces tumour regression as a monotherapy in vivo, indicating that targeting miR-346:NORAD balance is a valid therapeutic strategy.
A balancing act between miR-346 and NORAD regulates DNA damage and repair in PC. miR-346 may be particularly effective as a therapeutic in the context of decreased NORAD observed in advanced PC, and in transcriptionally-hyperactive cancer cells.
Molecular cancer. 2022 Mar 22*** epublish ***
C E Fletcher, L Deng, F Orafidiya, W Yuan, M P G S Lorentzen, O W Cyran, A Varela-Carver, T A Constantin, D A Leach, F M Dobbs, I Figueiredo, B Gurel, E Parkes, D Bogdan, R R Pereira, S G Zhao, A Neeb, F Issa, J Hester, H Kudo, Y Liu, Y Philippou, R Bristow, K Knudsen, R J Bryant, F Y Feng, S H Reed, I G Mills, J de Bono, C L Bevan
Imperial Centre for Translational and Experimental Medicine, Department of Surgery & Cancer, Imperial College London, London, UK. ., Imperial Centre for Translational and Experimental Medicine, Department of Surgery & Cancer, Imperial College London, London, UK., Institute of Cancer Research and The Royal Marsden NHS Foundation Trust, Sutton, UK., Division of Cancer and Genetics, School of Medicine, Cardiff University, Heath Park, Cardiff, UK., Institute for Radiation Oncology, Department of Oncology, University of Oxford, London, UK., Translational Oncogenomics, Manchester Cancer Research Centre and Cancer Research UK Manchester Institute, Manchester, UK., Department of Human Oncology, University of Wisconsin School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI, USA., Transplantation Research and Immunology Group, Nuffield Department of Surgical Sciences, University of Oxford, Oxford, UK., Section of Pathology, Department of Metabolism, Digestion and Reproduction, Imperial College London, London, UK., Veracyte, Inc., San Diego, CA, USA., Nuffield Department of Surgical Sciences, University of Oxford, John Radcliffe Hospital, Oxford, UK., Department of Cancer Biology, Thomas Jefferson University, Philadelphia, PA, USA., Departments of Urology and Radiation Oncology, University of California San Francisco, San Francisco, CA, USA.